Visualizing phosphodiester-bond hydrolysis by an endonuclease.

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• Enzymatic hydrolysis of DNA phosphodiester bonds is crucial for DNA metabolism and repair.
• The precise chemical mechanism of DNA cleavage by endonucleases, particularly the role of metal ions, remains incompletely understood.

Purpose of the Study:

• To observe and characterize the chemical reaction mechanism of DNA double-strand break (DSB) generation by the Desulfurococcus mobilis homing endonuclease I-DmoI.
• To elucidate the role of metal ions in the catalytic process of DNA cleavage.

Main Methods:

• Time-resolved structural studies capturing sequential catalytic intermediates.
• X-ray crystallography to visualize protein-DNA-metal complexes at different reaction stages.

Main Results:

• Captured distinct intermediate states of the I-DmoI endonuclease during DNA cleavage.
• Observed the sequential binding of two metal ions and the crucial role of a third metal ion in triggering phosphodiester bond hydrolysis.
• Documented the orchestrated conformational changes in protein, DNA, and metal ions throughout catalysis.

Conclusions:

• The study provides the first direct observation of the chemical reaction mechanism for DNA DSB generation by I-DmoI.
• A three-metal-ion mechanism is proposed, where a transient third metal ion is essential for catalyzing the hydrolysis of DNA phosphodiester bonds.